Culture for butyric acid fermentation



3o lar forms seldom seen.

' slight; odor absent; sediment flaky Patented Nov; 28,1939

Rai'ael Arroyo, Rio

mesne assignments,

Rico

ATION Piedras, P. 3., assignor, by,

to Borlnquen' Associates, Inc., San Juan, P. R...

a corporation of Puerto No Drawing. Original application July 21, 1930,

Serial No. 92,934.

tion February 23, 1939, Serial No.

-Divided and this applica- 6 Claims. (01. 195-59) This invention relatesto the production of butyric acid by fermentation of carbohydrates, andis a division of my co-pending application, Serial No. 92,934, filedJuly 27, 1936; I

I It1has'already been proposed to make a mixture of products, includingnormal butyric acid,

by fermentation of glucose, but the yield was relatively low andinemcient, and the process was limited to the fermentation of hexoses,such as 10 glucose. I

' It has now been found that such manuiacture may be accomplished withahigh theoretical emciency, and with the yield oi. a large quantity forbutyric acid relative to the 18 bohydrates in thefermentable substrate,by employing a bacillus found on the seed of the annattb plant (Bimaorellana).

The following is a full description of the micro-organism utilized:Clostridium saccharo- 20 buturicum (new species).

I. Morphological characteristics Vegetative cells-0n dextrose agar, pH6.6, temp. 34 degrees 0., age 48 hours; size 1.0-1.5 by

with 24 hours; anaerobic growth,'being terminal and spherical withgreatly enlarged cells; irregu- Microscopically, cells in extremely longchains, as many as 59 having been observed in one chain.

11. Cultural characteristics 3 Agar colonies-Dextrose agar: pH 6.6,temp.

34 degrees 0.,-"age 48 hours: form circular; surface smooth; edgesentire;jelevation of growth convex; optically opaque.

Agar stroke.-Dextrose, pH 6.6, temp. .34 degrees 0., 24 hours: growthmoderate; luster dull; chromogenesis none; odor of butyric acid; mediumunchanged.

Nutrient broth.-pH 6.7, temp. 34 degrees 0., age 48 hours: surfacegrowth none; cloudiness and abundant.

Gelatin stab.-Temp.'32.5 degrees 0., age 48 hours; growth best at top;liquefaction stratiiorm beginning in 12 hours complete in less than 48hours. Standard methods used, medium unchanged.

Temperature .relations.-In molasses wort, pH 6.8-7.0, optimum -34degrees C. Chrom0genesis.-None on any media. I AcidityP-Optimum pH6.8-7.0. Oxygen requirements.-Pyrogallol and potassium hydroxide used,\mediumcdextrose agar, temp. 34 degrees 0.: 60 better than aerobic.

aerobic absent, anaerobic total available carg 5.6-8.4 microns inlength,'ends rounded, slightly Mills-34 degrees 0., I 96 hours: acidcurd; slightpeptonization.

Litmus milk-34 degrees 0.: litmus reduction begins in 24 hours; complete48 hours; no-curd; no peptonization; gas; slight acid.

Indole tryptophane broth-34 degrees 0., 96 .hours; negative.

Yeast water extract sulfite cyan-pH 7.0, 34

degrees 0.; very positive, 48 hours.

Nitrates, nitrate broth-34 degrees 0.; growths scanty, no nitrite or gasin 96 hours.

Indicator reduction-Medium milk, pH 6.8, 34 degrees 0.: Janusgreenreduced 48 hours; methylene blue reduced 24 hours: litmus reduced48 hours.

Staining-Gram positive (Kopelloflf Beemans modification) Sporespositive, Ziehl Nielsen. Stains fairly easily with most stains.

I -Flaoella.0apsules absent.

m. P ysiological characteristics Starch hydrOZysisF-Standard methods,media aerobic and anaerobic, negative 96 hours.

Nutrient agar, with 2 percent soluble starch, slight hydrolysis in 72hours aerobically.

Egg albumen-Not blackened or liquefied. Brain-Not blackened. v pDextrose nutrient bile, pH 7.2,. 34 degrees 0., 48 hours: iair growth;stringy sediment, medium unchanged.

. IV. Fermentation reactions With carbohydrates, bacto-nutrient broth,with indicator, at 34 degrees 0.

Rotation Days raau'ua'aaa Mam! IEI"EEEEENI 1 xxxxVery abundant.

The organism occurs on the seed of the annatto plant (Bixa orcllana)which is indigenous to Puerto Rico. I'he seedis introduced into a flaskof sterile wort which may be comprised of a mash as described herein.After incubation of the flask and after fermentation has begun,transfers are made to a second flask of sterile 5 Wort, and the growthallowed to continue.

One method of employing the organism is toprepare a mash comprising 1part of black strap molasses, by weight, to 7 parts of water, and mixingthoroughly, 0.135 part by weight of calcium 10 carbonate is then added.The mash then has a density of approximately 10.0 Brix, and issterilized in an autoclave at 10 pounds pressurefor 30 minutes. Theacidity is adjusted to the preferential range of pH 6.8-7.00, and themash is seeded with approximately 10 percent by volume of inoculatingmaterial in the form of an actively fermenting culture (18-24 hours old)of the organism, and permitted to ferment in a non-corrosive vessel(such as wood or glass) at 20 the optimum temperature of 30 to 32degrees 0.,

for a time of about 96 hours.

After fermentation is substantially completed, the mash is tested withlitmus and any free acid neutralized with calcium carbonate. The

neutral or slightly alkaline mash is then evaporated down to one-fourthof its original volume, preferably under vacuum. The thickened orpartially evaporated mash is then treated with sulphuric acid toseparate the fatty acid from its calcium salt, and the mixture is thensteamdistilled until all of the fatty acid has passed over into thedistillate. This distillate is a .dilute mixture of acid and water, andis then treated with barium hydroxid, and the butyric 35 acid separatedand recovered as the barium salt.

The product comprises 46 to 47 percent of butyric acid on the basis ofthe total sugars in the molasses mash, equivalent to 2.50 to 2.75 poundsof butyric acid per gallon of blackstrap molasses fermented with atheoretical efllciency of 96.0 to 97.5 and a 3 percent concentration ofacids calculated on the weight of the mash.

Of the acids formed by the fermentation, butyric acid constitutes about99 percent or more of the total acids. in traces only, and analysesindicate that the yield of precipitated barium salt is comprised ofalmost 100 percent of pure butyrate. The ratio of gases-formed by volumeof hydrogen to carbon dioxide is about 7:4, and by weight is about45:56.3. The ratio if total gas to acid is about 781.2 cc. per gram ofacid formed.

It has been found that the organism is capable of fermenting sucrose asreadily as invert sugars,

and hence there is no necessity for inverting the mash beforeinoculation. The time required for the completion of fermentation, andthe final.

efliciency of conversion of the sugar, is as satisfactorywhere allsucrose is used'as where the fermentable carbohydrates consistedexclusively of hexose sugars. Further, it will be noted that excellentfermentations can be produced of the carbohydrates glucose,fructose,'galactose, mannose, sucrose, maltose,and of inulin, glyceroland Other acids are thus present 1 ammonium sulphate.

grees C., preference being given to a temperature of, 30 to 32 degreesC. At successive temperaures higher than 34 degrees, it is found thatthe production of acid falls off sharply and that. at 40 degrees, theyield is considerably lower than 28 degrees C. The efficiency is greaterat the preferred temperatures of 30 to 32 degrees C., and the timerequired for the completion of the fermentation is materially shorterthan at the lower temperatures within the bracket of 28 to 40 degrees C.

In fixing the sugar concentration, it has been found that the optimumcomprises from 4.59 to 8.52 percent of sugars in the solution, with thepreferred concentration around 6.5 percent, as this is a satisfactorilyhigh density, both of the sugar and of electrolytes in the subtrata, andalso the toxic effects of the products of metabolism do notdeleteriously affect the continued propagation of the biochemical agentsduring thecourse of fermentation.

It has been found that, in operating for extended periods, it isadvantageous to employ noncorrosive material for the fermentation vat.Wood and glass have been found satisfactory. When using fermenters withexposed surfaces of metals toxic to the organism, it has been found thatthe products formed have a very detrimental corrosive effect upon themetal itself, and that the dissolved metal proves very detrimental tothe yield of products of metabolism, and to the propagation of theorganism. Fermenters of bare copper metal could not be used; and withiron vessels, the progress was attended with much impaired efliciency.It was found that an enameled iron was not fully satisfactory, as theenamel did not protect the organism from the toxic action of dissolvedmetal, nor did it protect the iron from the corrosive effects of theproducts of the fermentation.

It is preferred to keep the surface-volume ratio as low as possible, ashydrogen is liberated during the course of the fermentation; and a lowratio has a "considerable influence in determining the efilciency of theconversion .of sugars into the desired products. A minimum surfaceexposure is recommended.

When molasses is employed as a base for the fermentation mash, it isnecessary to make sure that there is nonutrient deficiency. The elementmost frequently found to be lacking is nitrogen. Cuban molasses maycontain from 1.25 to 1.5 percent'of nitrogen; Puerto Rican molassesaround 1:00 percent, and Egyptian mo-. lasses around 0.3 to 0.5 percent.Such nitrogen deficiency may be made up by ammonium or nitrogen oxidecompounds, such as ammonium carbonate, but it is important to avoid theuse of In testing the addition of ammonium sulphate with standard mashesover a range of from 0.6 to 1.25 grams per liter,

it was 'found that this particular nitrogenous material ,wasdetrimental, as hydrogen sulphide Was formed and the yield of aciddecreased. It is therefore'recommended to avoid the addition ofsulphur-containing materials to the mash.

More 'rarely, it has been found that phosphorous is deficient; and inrare instances,

,manganese and even copper have been found lacking in proper proportion.

The course of the fermentation of the mash r is promptly evidenced bythe vigorousevolution of gas, and the bleaching of the color of the mashdue to the increasing acid reaction. The fermentation proceeds at anaccelerated rate for of the pH value with the excess of the calciumcarbonate is sufficient for the course of the fermentation.

Further, the production of butyric acid by the present fermentationmethod is excellently adapted for commercial employment, as it avoidsthe usual high costs of purification of the end products offermentation. The efliciency of production of the final acid is veryhigh, and this final acid product consists almost entirely of butyricacid. Hence the usual stages of refractlonation and purification of aheterogeneous mixture is avoided, as the acid can be distilled in apractically pure condition from the fermented mass. By employing theOthmer method, (Chemical and Metallurgical Engineering December, 1933,page to dehydrate and distill simultaneously, and the butyric acid maybe recovered as such without passing through the form of metalbutyrates. f-

The organism has been found excellently adapted for employment withblackstrap molasses of thetype madein Puerto Rico and Cuba as finalsugar house molasses, and containing the usual percentages of sugarstherein. The organism also works with equal efficiency on beet finalmolasses, when this is employed in preparing the substrate; and also, asset out above,

other substrata may be utilized therewith.'

631 et seq.), it is possible I claim:

1. A butyric acid fermentation mash of Clostridium saccharobutyricum andan aqueous substrate having a concentration of substantially 6.5 percentby way of sucrose and invert sugars.

2. A butyric acid fermentation mash of Clostridium sacchdrobutyricum andan aqueous substrate having a concentration of 4.59 to 8.52 percent ofacompound selected from the group consisting of glucose, fructose,galactose, man nose, sucrose, maltose, inulin, glycerol, and mannitol.

3. A butyric acid fermentation mash of Clostridium saccharobutwicum andan aqueous substrate having a concentration of 4.59 to 8.52 percent of acompound selected from the group consisting of glucose, fructose,galactose, mannose, sucrose, maltose, inulln, glycerol and mannitol andhaving a pH value of 6.8 to 7.0.

4. A butyric acid fermentation mash of Clostrz'dium saccharobutyncum anda dilute sterilized molasses solution and having an acidity ofsubstantially pH 6.8 to 7.0.

5. A butyricacid fermentation mash of Clostridium saccharobuturicum anda 'dilute molasses solution, and containing an excess of calciumcarbonate over that required for neutralizing acid present.

6. A butyric acid fermentation mash of Clostridium sacchdrobutyricum anda dilute blackstrap molasses solution having a total sugar concentrationof 4.59 to 8.52 percent, and a pH of substantially 6.8 to 7.0 andcontaining excess calcium carbonate.

- RAFAEL ARROYO.

